Antibody-based therapeutics, i.e., monoclonal antibodies (mAbs) and Fc fusion proteins, have now “come of age” as therapeutics. There are at least eighteen mAbs and two fusion molecules on the market and more than 150 are currently in clinical trials (see, e.g., Holliger et al. (2005) Nature Biotech., 23:1126-1136 and Theillaud (2005) Expert Opin. Biol. Ther., 5(Suppl. 1):S15-S27). Indications for these therapeutics are varied and include, e.g., organ transplantation (OKT3®, Simulect®, Zenapax®), oncology (Rituxan®, Panorex®, Herceptin®, Mylotarg®, Campath®, Zenapax®, Bexxar®, Erbitux®, Avastin®, HuMax-CD4™), infectious disease (Synagis®), inflammation and autoimmune disease (Humira®, Amevive®, Enbrel®), and allergic asthma (Xolair®). The therapeutic activity of such drugs may be mediated via different mechanisms of action, for example, by inhibiting signaling events in target cells, by direct induction of apoptosis, as well as by indirect immunologic mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to Fc receptors and complement-dependent cytotoxicity through binding to C1q (both mechanisms are termed collectively as “effector functions”).
Mouse mAbs were first made by Köhler et al. in 1975 (Nature (1975) 256:495-497). The first mAb that was approved for clinical use is a murine antibody (OKT3®). However, the effector functions, immunogenicity, and the pharmacokinetic properties of mouse antibodies (most of them being IgG1 or IgG2a and, in some cases, IgG2b) are generally not satisfactory for therapeutic uses in humans. For example, when mouse antibodies are tested with cells of human origin, the level of ADCC is substantially lower than that with mouse cells. Further studies elucidated that the antibody Fc regions are responsible for effector functions, and that the reduced ADCC is due to a lower binding affinity of murine IgG Fc region to human Fcγ receptors as compared to human antibodies.
Much effort has been made to produce antibody-based therapeutics with decreased immunogenicity and optimized effector functions in humans. As a result, chimeric, humanized and fully human monoclonal antibodies and antibody-based fusion proteins have been developed. Most chimeric and humanized antibodies, as well as antibody-based fusion molecules, contain an Fc region derived from human IgG1, because this subclass exhibits characteristics (FcγRs binding, serum half-life) and functional properties (ADCC, phagocytosis, endocytosis, complement activation) desirable for certain types of immune intervention.
Although some antibody-based therapeutics may function without utilizing antibody effector mechanisms, others may need to recruit the immune system to kill the target cells. If immune system recruitment is desirable for a particular therapeutic, engineering the IgG Fc portion to improve effector function (e.g., improved binding to IgG receptors and/or complement) may be a valuable enhancement.
Several strategies have been explored to enhance immune system recruitment, including: bispecific antibodies, in which one arm of the antibody binds to an Fcγ receptor (see, e.g., Segal et al. (1999) Curr. Opin. Immunol., 11:558-562); cytokine-IgG fusion molecules (e.g., IL-10-Fc, IL-15-Fc); and mutation of amino acid residues responsible for binding to FcγRs (see, e.g., Shields et al. (2001) J. Biol. Chem., 276:6591-6604).
Glycosylation of immunoglobulins can be an essential determinant of effector functions. Therefore, another approach to modify the effector function of a particular IgG is to engineer the glycosylation pattern of the Fc region.
An IgG molecule contains an N-linked oligosaccharide covalently attached at the conserved Asn297 of each of the CH2 domains in the Fc region. The oligosaccharides found in the Fc region of serum IgGs are mostly biantennary glycans of the complex type. Variations of IgG glycosylation patterns include attachment of terminal sialic acid, a third GlcNAc arm (bisecting GlcNAc), a terminal galactosylation, and α-1,6-linked core fucosylation. Oligosaccharides can contain zero (G0), one (G1), or two (G2) galactoses (see FIG. 1A). The exact pattern of glycosylation depends on the structural properties of IgG subcomponents, in particular, CH2 and CH3 domains (Lund et al. (2000) Eur. J. Biochem., 267:7246-7257). The cell lines used to produce recombinant IgG mAbs or fusion molecules (most often derived from mouse and hamster cell lines) may also influence the synthesis of oligosaccharide chains.
The oligosaccharide moiety of glycoproteins is initially biosynthesized from lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol which is then transferred to the protein in the endoplasmic reticulum (ER) (see FIG. 1B). The oligosaccharide portion is then processed in the following sequence. First, all three glucose (Glc) residues are removed by glucosidases I and II to yield Man9GlcNAc2-protein. The Man9GlcNAc2 structure may be further processed by the removal of a number of mannose (Man) residues. Initially, four α1,2-linked mannoses are removed to give a Man5GlcNAc2-protein which is then lengthened by the addition of a N-acetylglucosamine (GlcNAc) residue. This new structure, the GlcNAcMan5GlcNAc2-protein, is the substrate for mannosidase II which removes the α1,3- and α1,6-linked mannoses. Thereafter, the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of structures often found on glycoproteins.
Several studies have investigated the relationship between IgG glycoforms and FcγRIII-dependent ADCC.
Galactose—Removal of most of the galactose residues from a humanized mAb IgG1 (Campath®) resulted in reduced complement lysis activity but had no effect on ADCC (Boyd et al. (1995) Mol. Immunol., 32:1311-1318). However, a highly galactosylated form of a human anti-RhD monoclonal IgG is more active in ADCC assays than the agalactosyl form (Kumpel et al. (1994) Antibodies Hybridomas, 5:143-151). Thus, the impact of galactosylation of IgG oligosaccharide on ADCC is controversial.
Static Acid—The terminal sialic acid seems to have no effect on ADCC (Boyd et al. (1995) Mol. Immunol., 32:1311-1318).
N-acetyl-glucosamine—Several studies have focused on the role of bisecting GlcNAc in binding to FcγRIII and ADCC. The glycosylation pattern of a chimeric IgG1 antineuroblastoma antibody has been engineered in CHO cells transfected with β-1,4-N-acetylglucosaminyltransferase III (GnTIII) (Umana et al. (1999) Nature Biotech., 17:176-180; see also U.S. Pat. No. 6,602,684). This enzyme catalyzes the addition of bisecting GlcNAc residue to the N-linked oligosaccharide. The bisecting GlcNAc blocks the α-1,6-linked core fucosylation of N-glycans, since α1,6-fucosyltransferase cannot efficiently use bisecting N-glycans as substrates (Longmore et al. (1982) Carbohydrate Res., 100:365-392). IgG produced in this cell line exhibited an increased ADCC activity. However, the contribution of bisecting GlcNAc on effector functions as compared to core fucose remains controversial (Shinkawa et al. (2003) J. Biol. Chem., 278:3466-3473).
Fucose—Humanized and chimeric IgG1 mAbs have been produced in a rat hybridoma cell line that expresses a lower level of α-1,6-fucosyltransferase, so that the secreted mAbs have lower fucosylated oligosaccharide than Chinese hamster ovary (CHO)-produced IgG1 (Shinkawa et al. (2003) J. Biol. Chem., 278:3466-3473; see also European Patent Appln. Pub. No. 1176195). These studies have shown that non fucosylated oligosaccharides play a more critical role in enhancing ADCC than bisecting GlcNAc oligosaccharides. This report is consistent with previous studies in which the fucose deficiency of IgG1 had no effect on C1q binding, but provoked an increased binding to human FcγRIIIA and allowed a higher ADCC activity (Shields et al. (2002) J. Biol. Chem., 277:26733-26740).
Attempts have been made to engineer cell lines that produce recombinant IgG with a well-defined pattern of glycosylation in the Fc region. For example, CHO cell lines expressing high levels of human β-1,4-galactosyltransferase (GT) and/or α-2,3-sialyltransferase (ST) have been made. The structure of IgG oligosaccharides produced in these cells shows a greater homogeneity as compared with control cell lines. Overexpression of GT reduces the amount of terminal GlcNAc, whereas overexpression of ST increases sialylation of oligosaccharides (Weikert et al. (1999) Nature Biotech., 17:116-1121).
There continues to be a need to optimize antibody-based therapeutics, and in particular, to develop methods for producing antibody-based therapeutics with enhanced ADCC activity.